A Semi-throughput Procedure for Assaying Plant NADP-malate Dehydrogenase Activity Using a Plate Reader.

ABSTRACT

Chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a redox regulated enzyme playing an important role in plant redox homeostasis. Leaf NADP-MDH activation level is considered a proxy for the chloroplast redox status. NADP-MDH enzyme activity is commonly assayed spectrophotometrically by following oxaloacetate-dependent NADPH oxidation at 340 nm. We have developed a plate-adapted protocol to monitor NADP-MDH activity allowing faster data production and lower reagent consumption compared to the classic cuvette format of a spectrophotometer. We provide a detailed procedure to assay NADP-MDH activity and measure the enzyme activation state in purified protein preparations or in leaf extracts. This protocol is provided together with a semi-automatized data analysis procedure using an R script.